Integron and its role in antimicrobial resistance: A literature review on some bacterial pathogens

In recent years, different acquired resistance mechanisms, including transposons, bacteriophages, plasmids, and integrons have been identified as involved in the spread of resistance genes in bacteria. The role of integrons as mobile genetic elements playing a central role in antibiotic resistance has been well studied and documented. Integrons are the ancient structures that mediate the evolution of bacteria by acquiring, storing, disposing, and resorting to the reading frameworks in gene cassettes. The term integron describes a large family of genetic elements, all of which are able to capture gene cassettes. Integrons were classified into three important classes based on integrase intI gene sequence. Integrons can carry and spread the antibiotic resistance genes among bacteria and are among the most significant routes of distribution of resistance genes via horizontal transfer. All integrons have three essential core features. The first feature is intI, the second one is an integron-associated recombination site, attI, and an integron-associated promoter, Pc, is the last feature. Among them, the class 1 integron is a major player in the dissemination of antibiotic resistance genes across pathogens and commensals. Various classes of integrons possessing a wide variety of gene cassettes are distributed in bacteria throughout the world. This review thus focuses on the distribution of integrons among important bacteria.


Introduction
Integrons are the ancient structures that mediate the evolution of bacteria by acquiring, storing, disposing, and resorting to the reading frameworks in mobile elements called cassettes. They are present in approximately 17% of the bacterial chromosomes (1). These structures are found in different environments such as forest, desert soils, river sediments, antarctic soils, hot springs, biofilms, plant surfaces, marine sediments, and deepsea sediments. Nowadays, the term integron describes a large family of genetic elements, all of which are able to capture gene cassettes. Although about one-third of integrons have been found in bacterial genome that do not carry gene cassettes (empty integrons) (2). Although the first antibiotic-resistant bacteria were reported in the mid-1950s, until the 1970s it was not clear that resistance phenotypes were associated with transmissible plasmids or elements. In the late 1980s, integrons were identified. Integrons play an important role in the distribution of antibiotic resistance, especially in Gram-negative pathogens. In resistance integrons, an action plan is associated with genetic moving elements such as transposons or plasmids, so interspecies and intraspecies transmission are increased. It has now been well established that integrons act as the main reason for multiple resistance in Gram negative more than in Gram-positive bacteria (3).

Classification of integrons
Integrons can be discriminated based on the relative homology of intI, although the cutoff point is not clear (2). Initially, it was suggested that integrons can be divided into two categories: 1) mobile integrons: have a small number of cassettes, usually encode antibiotic resistance, have different attC sites and their mobility is dependent on transposons or plasmids, 2) super integrons: have many cassettes, homogenous attC sites and located on the chromosome (4). Also, phylogenetic studies showed that a branch of integrons is associated with organismal phylogeny. These integrons are divided into three groups based on the phylogeny of the integrase genes: (i) a group of isolated Proteobacteria from freshwater and soil, including clinical integrons in classes I and III. (ii) a group found in Gamma-Proteobacteria in marine environments that includes class II, SXT integrative conjugative elements integrons and pRSV1 plasmids in Vibrio; and (iii) the integrons whose integrase genes are inverted. These inverse integrons have been found in Spirochaetes, Planctomycetes, Cyanobacteria, and Chlorobi spp. (4). Up to now, more than 9 classes of integrons have been identified based on 16 amino acids conserved in Gram-negative bacteria (5), but only 4 main classes are associated with clinical isolates. As stated above, the amino acid sequencing of the integrase gene is used as a marker for the classification of integrons Integrons and antimicrobial resistance Sabbagh et al. into different classes, and members having the same integrase have the same class but can carry different gene cassettes. Of all the different classes of integrons, the class I integrons and then class II are the most common classes among clinical isolates (6). Classes I to III are also integrons with multiple resistance. The class IV integrons are considered a distinct type of integron called super integron, which is found on the small chromosome Vibrio cholera (7).

Integrons structure
Structurally, all integrons consist of three main components, including 5′ and 3' conserved segment and a central variable region between the 5' and 3' zone, in which integrons are responsible for the capture and expression of exogenous genes, which are part of the gene cassette (1). Essential components of the 5' and 3' zone in all integrons include 1) integrase gene (intI) encoding a specific recombinant site and a part of the family of tyrosine recombinase (8), 2) the attI receptor site identified by integrase and adjacent to the intI gene, so that attI is in the upstream of intI, except for the Triponama spp. (9). Also, integrase protein can catalyse a recombinant between the input gene cassette and the attI. 3) the promoter sequence consists of P c and P int which is placed inside intI or between intI and attI and causes expression of the existing genes in the integrated gene cassette in integrons and integrase (9). The 3' conserved segment of integrons has different structures that differ in integron classes. The gene cassettes are located between the 3' and 5' zone, where integrons receive new genes through these cassettes.
The integron system has two important benefits as a genetic innovation: the new genetic material is integrated into the bacteria genome at attI, therefore, they do not cause abnormalities in the existing genes; second, the new integrated genes are expressed by the integrons promoter (Pc) (Figure 1) (10).

Gene cassette
The number of cassettes reported differ from zero to 100 in different studies, this difference in the number of cassettes is seen in Vibrio cholerae and some other species of Vibrio. A great variety is created in integron classes through the acquisition of various gene cassettes (11). Many studies have been done on the origin of the gene cassettes, but so far with no result. However, the size and frequency of gene cassettes suggest that some organisms, using single or pair genes, can create a gene cassette, the mechanism of which method has not yet been identified. These cassettes are mobile elements that code multiple genes, including antibiotic resistance genes. These antibiotic classes include all known β-lactams, all aminoglycosides, chloramphenicol, trimethoprim, streptothricin, erythromycin, quinolones, rifampin, lincomycin, fosfomycin, and antiseptics of the quaternary ammonium-compound family (1,3). According to previous studies, gene cassettes are randomly combined in the region between the 3' and 5' conserved segment of integrons (12). The integration of gene cassettes is done with the integrase gene between two recombination sites (attI, attC) which process is reversible and cassettes can be released in the form of free DNA from integrons ( Figure 2) (11). In general, Figure1. Integron structure intI, a gene for the integron integrase; attI, recombination site; Pc, an integron promoter. Gene cassettes, sequentially inserted into an array via recombination between attI and the cassette associated recombination sites (attC).  (13). Gene cassettes lack the promoter sequences in their structure, thus expressing the gene cassettes associated with an external promoter (Pc) in the structure of integrons. Because integrons have promoter sequences, they can express the genes in the gene cassettes, so integrons act as both the expression vector of the gene and as a natural cloning system (12). There are areas at the end and beginning of a gene cassette that consist of a protected sequence including GTTRRRY (representing recombination activity). Although attC has distinct sizes between 57-141 bp, they have a set of common characteristics. These elements have a core site (CS) with the sequence GTTRRRY and an inverse core site (ICS) with the sequence RYYYAAC. The attC sequence was formerly known as 59bp element (14). All 59bp elements have a symmetrical central axis in their structure, on both sides of this axis of symmetry, there are ICS and CS sites, which has led to the creation of hairpins in the 59bp element. The attC sequences are categorized into different groups, based on the size of the first and largest group of 12 members and dependent on aacA(Iia), aadA, aadB, CatB3, and orfD genes. The factors present in these genes are only different in some base pairs (15).

Mobility
The mobility of integrons has been considered to be a major concern in clinical pathogens of spreading antibiotic resistance, and this mobility is related to mobile DNA elements (transposon and plasmid) (16). However, integrons do not have the ability to move, but integrons (mostly Class I) are known as mobile genetic elements that are most commonly found on transferable plasmids. Therefore, these moving plasmids carry gene cassettes that can transfer to other integrons or even to the bacterial genome. The integron system allows microorganisms to combine gene cassettes and convert them into functional proteins by expressing the genes correctly (2). Mobile genetic elements containing plasmids, transposons, secretion sequences, and genetic islands can act as extensive reservoirs of information for integrons, which are shared among bacteria. With the motion of the gene cassettes, the integrons play an important role in the distribution and spread of resistance genes. In addition to clinical aspects, there are many reports of the presence of integrons in environmental microorganisms, which indicate their high diversity in various functions. This explains exactly that integrons are old genetic elements within the genome and play an important role in evolution and adaptation (13).

Class I integron
In 1998, class I integron was found for the first time in Corynebacterium glutamicum, which is a Gram-positive microorganism (7), first discovered by Hall and Stoke in 1989 (17), and then it was observed in Corynebacterium, Streptococcus, Enterococcus, Staphylococcus, Aerococcus and Brevibacterium. Class I integron has the highest frequency among the integrons (11,18). IntI is able to identify three types of recombinant sites (attI, attC, and secondary sites). Therefore, these types of integrons are able to receive gene cassettes through the recombination of dedicated sites. Also, class I integron has a direct relationship with Tn402 and the Tn3 family of transposons (1,18). In this class of integrons, the gene cassettes can be further expressed through the promoters P c andP 2 (second promoter that is usually inactive). P c plays an important role in integron function because it ensures the correct expression of the gene cassettes. Class I integron has been studied in different microorganisms. The prevalence of class I integron is about 22 to 55%, among the Gram-negative bacteria isolated from the clinic, which include Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Campylobacter, Citrobacter, Enterobacter, Escherichia, Klebsiella, Mycobacterium, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Stenotrophomonas, and Vibrio (19), and it is also found in about 22 to 59% of Campylobacter jejuni, Providencia stuartii, Serratia marcescens, Stenotrophomonas maltophilia (7). Class I integron acts as a common factor in the distribution and spread of antimicrobial resistance. This class carries over 40 resistance genes related to aminoglycosides, betalactams, chloramphenicol, macrolides, sulfonamides, disinfectants, and dexophthane (20). The 3′-conserved segment in class I integron consists of the following components: 1) qacEΔ1 gene encoding resistance to quaternary ammonium salts and dexophthane, 2) sul1 gene encoding resistance to sulfonamides (14), 3) orf5 has no known function but is similar to puromycin acetyltransferase in Streptomyces albonier, this suggests that it leads to resistance to puromycin through the mechanism of acetyltransferase ( Figure 3) (21).

Class II integron
Class II integrons indicate a high prevalence in clinical isolates in Gram-negative bacteria such as Acinetobacter, Shigella, Salmonella, Pseudomonas (7). Class II integrons, similar to class I, are also associated with the Tn7 family of transposons (Tn7 and its derivatives such as Tn1825, Tn1826, and Tn4132) which carry the recombinant site attI2 and P c . The 3′-conserved segment includes The following cassettes are a part of the 3′conserved region and not mobile: sul1 gene encoding resistant to sulfonamides, qacEΔ encoding resistant to quaternary ammonium compounds, and orf5 unknown function 5 tns (tnsA, tnsB, tnsC, tnsD, and tnsD) genes that play a role in the transposon movement. Class II integrons contain gene cassettes including dfrA1 (dihydrofolate reductase), sat1 (streptothricin-acetyl transferase), and aadA1 (aminoglycoside adenyltransferase) which are resistant to trimethoprim, streptomycin, and streptomycin/spectinomycin, respectively. The ereA gene (erythromycin esterase) has also been found in the class II integron (7,16). The integrase gene in class II integrons is about 46% similar to the integrase gene in class I integrons. One of the most important differences between intI1 and intI2 is that the integrase gene in the class II integrons (intI2) is stopped early by the end codons (TAA), resulting in the 178 amino acid protein synthesis being deactivated. Therefore, class II integrons are weaker in moving gene cassettes than class I integrons. However, mutations in end codons lead to reactivity of these amino acids, which results in the activation of the integrase gene (3).

Class III integron
In 1993, class III integrons were first identified in Japan by Arakawa and colleagues in S. marcescens. These types of integrons rarely present in clinical specimens, they have been found in a small number of bacteria such as Acinetobacter spp., Alcaligenes, Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella spp. and S. marcescens (7). The 3′-conserved segment in these integrons is similar to that of class I integrons: it contains four genes, qacEΔ1, sul1, orf5, orf6, and their only difference is the lack of transcription genes. In these integrons, there are several gene cassettes including bla IMP-1 and aacA4 that encode metallo-lactamase enzymes and the aminoglycoside resistance gene (tobramycin), respectively. The aacA4 cassette was originally found in class I integrons but the bla IMP-1 cassette was first recognized in class III integrons, however due to the extensive recombination that occurred between class I and III integrons, this gene is also found in class I integrons and has been widely reported worldwide (22).

Class IV integron
In fact, class IV integrons are super integrons that were first detected in Vibriocholera. Mazel in 1998, for the first time, called them super integron (19). These integrons are seen in microorganisms including Vibrionaceae, Shewanella, Xanthomonas, Pseudomonas, and other proteobacteria. To date, class IV integrons have been found to carry gene cassettes imparting resistance to the antibiotic's chloramphenicol and fosfomycin (23).

Antibiotic resistance
In recent years, excessive consumption of antibiotics has led to increased antibiotic resistance. Genetic mutations and acquisition of resistance genes are involved in the development of resistance. One of the most important causes of the development and spread of antibiotic resistance of genes is the acquisition of resistance genes that occur through the horizontal transduction of the gene or genetic moving elements such as plasmids and transposons (24). These integrons share a collection of genetic cassettes most of which encode antibiotic resistance. In general, about 130 resistant genetic cassettes have been identified with varying patterns of codon and attC sites (3). Resistance integrals have several common features: they usually have motion, their cassette arrangements are short, and typically only carry antibiotic resistance genes. However, these common features in them are not inherent characteristics of the ancestors of integrons but they are created by strong selective pressure during the use of antibiotics by humans.

Integrons in various bacteria
Escherichia coli E. coli is known as the head of the large family of Enterobacteriaceae. Based on diversity in pathogenesis and clinical symptoms, the strains of E. coli are divided into two intestinal and extra-intestinal. E. coli is one of the most important causes of gastrointestinal and urinary tract infections in humans that has been resistant to a wide range of antibiotics over the past years (25). Acquiring mobile elements, including plasmids, transposons, and integrons among Gram-negative bacteria, it plays an important role in the development of antibiotic resistance (2). Class I integron was reported in 1973 (26). Common cassettes in this class of integron result in resistance to aminoglycosides (aadA1, aadA2, aadB, aadA5), trimethoprim (dfrA1, dfrA5, dfrA7,  dfrA12, dfrA17, dfrB2, dfrA1-gcuC, dfrA17-aadA5, dfrA1-aadA1, dfrA12-gcuF-aadA2), erythromycin (ere2) and broad-spectrum beta-lactams (ESBL) (bla OXA-101 -aac(6')-Ib) (27). Also, class II integron has been reported less frequently. This integron contains gene cassettes dfrA1-sat1-aadA1, dfrA1-sat2-aadA1 and estX-sat2-aadA1 (28). Also, recently, a new class II integron has been identified (29), which includes two gene cassettes. One of them is the dfrA14 gene. There are also reports available on the availability of Class III integrons that are commonly seen with class I and III integrons in this bacterium (30). (Table 1) in our study in 2015, we collected E. coli from clinical isolates of patients in the north of Iran, which showed 22% of E. coli isolates carried class I integron ( Table 2) (31).

Pseudomonas aeruginosa
P. aeruginosa is one of the most important factors in hospital infections, especially in burn patients and patients with cystic fibrosis. Due to increased MDR bacteria, treatment for this bacterium is difficult to find. Class I integron is an important factor for the development of antibiotic resistance and the emergence of MDR strains (41). This class of integrons includes some gene cassettes such a aadB and aadA2 (resistance to aminoglycosides), dfrA17-aadA5 and dfrA12-gcuF-aadA2 (resistance to trimethoprim), bla CARB-2 (resistance to carbenicillin) (27). Class II integrons in P. aeruginosa include dfrA1-sat1-aadA1 (Table 1) (16). Our studies in 2012 and 2013 on P. aeruginosa isolates showed 37-40% of isolates had the intI1 gene (Table 2) (41,42).

Enterococcus faecalis
E. faecalis is an intestinal flora and has been identified as one of the major causes of hospital infections, with a tendency to increase antibiotic resistant class I integrons; E. faecalis contains gene cassettes dfrA12-gcuF-aadA2 (resistance to trimethoprim) and aadA1a (resistance to aminoglycosides) (43). Class II integrons has the dfrA1-sat1-aadA1 cassettes (Table 1) (44).

Staphylococcus aureus
S. aureus is a Gram-positive microorganism which is now considered one of the most important causes of hospital infections (45). Quantitative studies have been done on integrons in Gram-positive bacteria. Class I integrons is more common in S. aureus and causes resistance to aminoglycosides (aadA1a and aadA2), trimethoprim (dfrA17-aadA5 and dfrA12-gcuF-aadA2) and chloramphenicol (aacA4-cmlA1)(

Conclusion
Integrons being capable of integrating, expressing, and disseminating gene cassettes, carrying resistance determinants, play a critical role in facilitating the MDR phenotype in these bacteria. The ability of integrons to acquire new cassettes and their ability to recombine cassette rows emphasizes the adaptation of their diversity in bacteria. Their ability to rapidly spread resistance phenotypes makes it important to consider what other integron-mediated traits, such as increased pathogenicity, virulence, or resistance to antimicrobials might impact human health in the future. If we can control integrons and cassette formation, we could use integrons as a platform for enzyme discovery and to construct novel biochemical pathways in antimicrobial resistance. So, knowledge about the prevalence of integrons and gene cassettes is helpful for the treatment and correct use of antibiotics.